The present invention, in some embodiments thereof, relates to isolated cell populations comprising mesenchymal progenitor cells (MPCs), and more particularly, but not exclusively, to methods of generating same and using same for producing and isolating extracellular matrix which can be used for preparation of implantable devices for tissue regeneration and/or repair.
The aim of regenerative medicine is to repair or replace damaged or diseased tissue in the human body. Cell therapy based upon stem and progenitor cells have many distinct advantages and offer tremendous potential for regenerative medicine. The multipotency and proliferative nature of stem cells makes them a more reliable cell source than terminally differentiated cells. Stem cells have additional advantage of being relatively more immune-compatible cells. In addition, stem cells can proliferate well on a supportive scaffold, and their cell fate can be further controlled and directed by their interactions with the synthetic scaffold (Lim S H and Mao H Q, 2009, Advanced Drug Delivery Reviews, 61: 1084-1096).
Adult mesenchymal stem cells (MSCs) derived from either bone marrow or adipose tissues are multipotent cells that can differentiate into various lineages including osteogenic, chondrogenic and adipogenic lineages, and as such can be used to enhance repair of a variety of soft tissue defects. However, adult MSCs exhibit a limited capacity to proliferate, loss of differentiation potential and reduced protective factors during ex-vivo expansion before possible therapeutic use. In addition, adult stem cells isolated from different subjects exhibit remarkable variability. Moreover, while isolation of adult MSCs from a human body involves invasive procedures, aging and aging-related disorders significantly impair the survival and differentiation capacity of MSCs, thus limiting their therapeutic efficacy. Due to these limitations, adult stem cells are not suitable for “off the shelf product” such as for production of extracellular matrix (ECM).
Mesenchymal stem cell-like cells can be generated from pluripotent stem cells capable of differentiation into cells of all three embryonic germ layers (i.e., endoderm, mesoderm and ectoderm) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In contrast to adult stem cells, pluripotent stem cells can potentially indefinitely proliferate, providing a large number of cells with specific characteristics needed for regenerative medicine protocols. However, the practical use of pluripotent stem cells as a source for ECM, requires the development of simple and efficient protocols to generate “easy to grow” cell populations that can produce functional ECM for regenerative medicine.
Harkness L et al. 2010 (“Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential”. Bone Sep 30. Epub ahead of print) describe the direct differentiation of hESCs into stroma-like cells which differentiate into the osteogenic lineage while producing a mineralized matrix and into the adipogenic lineage while producing fat drops.
Hwang N S et al. 2008 (PNAS 105: 20641-20646) describe derivation of hESCs to MSCs capable of producing fat, cartilage and bone in vitro.
Induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed into a pluripotent state resembling that of human embryonic stem cells (hESCs). Patient-specific iPSCs can provide useful platforms for the discovery of new drugs, as well as unprecedented insights into disease mechanisms that ultimately may be used to develop cell and tissue replacement therapies (Kiskinis E, and Eggan K. J. Clin. Investigation, 120: 51-59, 2010). Human iPSCs (hiPSCs) have been generated from various types of somatic cells, most commonly fibroblasts that are isolated from tissues harvested via surgical intervention.
Novak et al., 2010 [Cell Reprogram. 2010, 12(6): 665-78)] describe the derivation of hiPSCs from plucked human hair follicle keratinocytes (HFKTs) which spontaneously differentiated into functional cardiomyocytes (CMs).
Lian Q et al., 2010 (Circulation. 121:1113-1123) have recently demonstrated the differentiation potential of hiPSCs into functional MSCs, using single cell sorting of CD105+/CD24− of differentiating hiPSCs. The resulting MSCs were capable of differentiation into adipogenic, osteogenic and chondrogenic lineages.
Li F. et al., 2010 (JCB 109: 643-652), describe the differentiation of murine iPSCs towards MSC-like cells by treating iPSC-derived-EBs with transforming growth factor beta-1 (TGFβ1) and retinoic acid. The resulting cells expressed putative MSCs markers and deposited calcium in vitro when cultured in an osteogenic medium.
WO/2007/080590 provides methods of generating multipotent connective tissue progenitor cells (CTPs) from embryonic stem cells and embryoid bodies. The CTPs population included 40-60% of CD105-positive cells.
WO/2007/080591 provides methods of generating multipotent connective tissue progenitor cells (CTPs) from adult stem cells.
The extracellular matrix (ECM) is a secreted product of cells that populate in a given tissue or organ. The ECM influences the behavior and phenotype of the resident cells. Cell attachment, migration, proliferation and three-dimensional arrangement are strongly affected by matrix composition and structure. The main advantages of using ECM scaffolds are their bioactivity and biocompatibility capabilities.
ECM composition includes the most abundant protein—type I Collagen, as well as fibronectin and laminin. Other substantial components are glycosaminoglycans, as chrondrotin sulfate, heparin and hyaluronic acid, which have superior binding properties of bioactive molecules as growth factors and cytokines. Growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and TGFβ, are present within the ECM in very small quantities but play a critical role as potent modulators of cell behavior. Individual components of the ECM, such as collagen I or fibronectin have been used as alternative scaffold materials, but were found to be less bioactive than the whole intact ECM.
ECMs for clinical applications are currently derived from organs such as the small intestine, urinary bladder or skin (Reing J., et al. 2009, Tissue Engineering, Vol. 15: 605-614′ Badylak S F. 2004. Transplant Immunology 12: 367-377), from allogeneic (human cadavers) or xenogeneic sources (porcine, bovine or equine small intestine submucosa, dermis and pericardium). Both cellular and acellular forms of ECM scaffolds have been used for tissue engineering applications. While the cellular form requires an autologous cell source, which is limited and may lead to patient's diminished functionality, the acellular form requires tissue processing, including elimination of all intact cells and degradation of nucleic acids, leaving only the ECM's proteins and growth factors biologically active. An example of a biological scaffold made of human cadaver is the GraftJacket™ product (Wright Medical Technology, TN, USA).
The ultimate goal in decellularizing a tissue, composed predominantly of Collagen fibers, is to remove any non-collagen components that may cause host rejection. However, in many cases the decellularization process is not complete and the scaffold includes traces of animal compounds, which elicit a significant inflammation response. In addition, cadaver donors are limited, thus scaffold made of this source are non-homogeneous, and non-reproducible. In addition, since derived from a human source, these scaffolds still exhibit the risk of pathogen transfer.
ECM mineralization is a physiologic process in bone, teeth, and hypertrophic cartilage, whereas in other locations it must be inhibited. Mineralization imparts important biomechanical and other functional properties to bones and teeth.
Synthetic scaffolds are manufactured from chemical compounds such as polyester, polypropylene, dacron, silicon and nylon fabric. Although they possess superior mechanical characteristics, they can never be integrated into the host tissue, and their poor biocompatibility causes numerous long-term complications, such as severe infections, chronic immune response and potential toxic byproducts (Chen J. et al. Expert Rev. Med. Devices, 2009, 6: 61-73).
Electrospinning can produce a macroporous scaffold comprising randomly oriented or aligned nanofibers. Electrospun polymeric fibrous meshes also offer a higher surface area for cell attachment and are relatively reproducible (Lim S H and Mao H Q, 2009; Adv. Drug. Del. Rev. 61:1084-1096).
Thibault R A et al. (Tissue Engineering, 2010, 16: 431-440) describe generation of a decellularized mineralized matrix from electrospun PCL fiber mesh scaffolds which were seeded with rat mesenchymal stem cells (MSCs) and cultured in a complete osteogenic medium.
WO/2009/098698 describes scaffolds composed of extracts of cellular and/or extracellular compartments for use in tissue regeneration.
The challenge in any reconstructive procedure is to provide a supporting structure while restoring the normal anatomic condition of the surrounding tissues. Though several materials can potentially provide the mechanical support, they do not possess the properties necessary to restore the living tissue's original quality.
Abdominal ventral hernia and pelvic floor defect (PFD) are common and challenging conditions for surgeons. It is estimated that 250,000 hernia repair and 300,000 procedures of prolapse and urinary incontinence surgeries are performed each year in the US. However, in about 12.5% of the hernia repair and 29% of the pelvic prolapse repairs repeated surgeries are needed within 5 years of initial surgery, mainly due to infection, seroma, wound dehiscence and formation of enterocutaneous fistula.
Synthetic meshes made of polypropylene and polyester are used for reconstructive surgeries (e.g., the Gyncare Prolift, Ethicon®, Johnson & Johnson, USA). Although the synthetic meshes are available and can simplify the operative procedure, reduce patient discomfort from an additional incision site and decrease operative time, in about 2.8-17.3% of the cases these meshes cause foreign-body reaction with risks of infection, rejection, visceral adhesion to the repair site, erosion to the bowel, urinary bladder and vaginal mucosa leading to enterocutaneous fistula, bowel obstruction and urinary bladder complications, extrusion of the repair material and infection. Infected synthetic repair material often necessitates surgical removal, leaving a contaminated field and a hernia deficit larger than the original (van't Riet M, et al., 2007. Hernia. 11:409-13; de Vries Reilingh T S, et al., 2007, World J. Surg. 31:756-63).
Additional background art includes Chin M H, et al., 2009 (CELL STEM CELL 5: 111-123]; Hu Q., et al., 2010 [Stem cells (ahead of print)]; Badylak S F et al. 2009 [ActaBiomaterialia, 5: 1-13]; Badylak S F 2004 [Transplant Immunology, 12: 367-377]; Cohen S. et al. [Tissue Eng Part A. 2010 (10):3119-37]; Shen J. et al. 2010 (Int J Artif Organs, 33: 161-170); Barbero A., et al., Arthritis & Rheumatism, 48: 1315-1325, 2003; Bieback K., et al., Stem Cells 2004, 22:625-634; U.S. Patent Application No. 20100185219 (to Arthur A. Gertzman et al.).